In the late 1930s, Oxford's Howard Florey and Ernst Chain happened across Fleming's report, and began to pursue studies to determine to true potential of penicillin. The pair was able to successfully isolate and purify penicillium notatum (growing their cultures in as unlikely containers as bedpans for want of laboratory dishes), and by 1941 had proved that it was an effective cure for bacterial infections. In 1943, New York physician John Friend Mahoney had identified the drug as a cure for syphilis and other infections.

After Florey and Chain took their findings to American pharmaceutical firm Eli Lilly and Company, mass production of penicillin began—though not in time to save the hundreds of soldiers that died from infections during World War II. Russian-born soil microbiologist Selman Waksman's discovery of streptomycin in 1944 marked the first effective medicine to treat mycobacterium tuberculosis (TB). Also providing relief for genito-urinary tract TB, the drug improved the outlook immeasurably when combined with synthetic isoniazid and para-amino salicyclic acid.